A CRISPR/Cas9 eraser strategy for contamination‐free PCR end‐point detection. Issue 5 (1st March 2021)

• Record Type: Journal Article
• Authors: Lin, Wei; Tian, Tian; Jiang, Yongzhong; Xiong, Erhu; Zhu, Debin; Zhou, Xiaoming
• Is Part Of: Biotechnology and bioengineering. Volume 118:Issue 5(2021)
• Page Start: 2053
• Page End: 2066
• Publication Date: 2021-03-01
• Subjects: contamination‐free -- CRISPR/Cas9 -- end‐point detection -- PCR -- RT‐PCR; Biotechnology -- Periodicals; Bioengineering -- Periodicals; 660.6
• DOI: 10.1002/bit.27718
• Languages: English
• ISSNs: 0006-3592

Abstract

Abstract: Polymerase chain reaction (PCR), a central technology for molecular diagnostics, is highly sensitive but susceptible to the risk of false positives caused by aerosol contamination, especially when an end‐point detection mode is applied. Here, we proposed a solution by designing a clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 eraser strategy for eliminating potential contamination amplification. The CRISPR/Cas9 engineered eraser is firstly adopted into artpcr reverse‐transcription PCR (RT‐PCR) system to achieve contamination‐free RNA detection. Subsequently, we extended this CRISPR/Cas9 eraser to the PCR system. We engineered conventional PCR primers to enable the amplified products to contain an implanted NGG (protospacer adjacent motif, PAM) site, which is used as a code for specific CRISPR/Cas9 recognition. Pre‐incubation of Cas9/sgRNA with PCR mix leads to a selective cleavage of contamination amplicons, thus only the template DNA is amplified. The developed CRISPR/Cas9 eraser, adopted by both RT‐PCR and PCR systems, showed high‐fidelity detection of SARS‐CoV‐2 and African swine fever virus with a convenient strip test. Abstract : A novel strategy combining CRISPR/Cas9 with conventional RT‐PCR and PCR was developed to construct a contamination‐free amplification, termed CRISPR/Cas9 eraser. Wei Lin and coworkers used a combination of RT‐PCR/PCR system, CRISPR recognition, and lateral flow test strip strategy to detect clinical SARS‐CoV‐2 samples and pig blood samples from suspected ASFV infection. They demonstrated the reliability, accuracy and convenience of this method and its potential as a more convenient molecular diagnostic technology without worrying the contamination and expensive detection device.